An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase,a key enzyme in artemisinin biosynthesis

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg²⁺ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519–532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg²⁺. Such detailed understanding will probably help to design an improved enzyme.     

Effect of Probiotic Soy Milk on Serum Levels of Adiponectin,Inflammatory Mediators,Lipid Profile,and Fasting Blood Glucose Among Patients with Type II Diabetes Mellitus

Probiotic therapies are going to be an effective alternative therapeutic strategy in the treatment and management of diabetes. The mechanism behind the essential effects of probiotic therapies in diabetic patients was not fully understood. The objective of this study was to evaluate the effects of probiotic soy milk containing Lactobacillus planetarum A7 on inflammation, lipid profile, fasting blood glucose, and serum adiponectin among patients with type 2 diabetes mellitus. Forty patients with type 2 diabetes, at the age of 35–68 years old, were assigned to two groups in this randomized, double-blind, controlled clinical trial. The patients in the intervention group consumed 200 ml/day of probiotic soy milk containing L. planetarum A7 and those in control group consumed 200 ml/day of pure soy milk for 8 weeks. Serum TNF-α, C reactive protein, adiponectin, lipid profile, and fasting blood glucose were determined before and after intervention. In intervention group, serum adiponectin in pre- and post-treatment did not show any significant changes (2.52 ± 0.74 vs 2.84 ± 0.61, P = 0.658), as well as changes in serum TNF-α and C reactive protein (172.44 ± 5.7 vs 172.83 ± 7.6, P = 0.278, 4.2 ± 1.4 vs 4.5 ± 1.9, P = 0.765, respectively). Low-density cholesterol and high-density cholesterol changed significantly (P = 0.023, P = 0.017, respectively), but fasting blood glucose did not show any significant changes. The results of this study showed that consumption of probiotic soy milk and soy milk has no effect on serum adiponectin and inflammation, but it can change lipid profile among type 2 diabetic patients.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

Simultaneous Cellulose Degradation and Electricity Production by Enterobacter cloacae in a Microbial Fuel Cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047^T based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m², compared to 5.4 ± 0.3 mW/m² for strain ATCC 13047^T. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.Exoelectrogenic microorganisms can release electrons to electron acceptors outside the cell, such as iron oxides or carbon anodes in microbial fuel cells (MFCs). Members of many genera, including Rhodoferax (6), Shewanella (13, 14), Pseudomonas (29), Aeromonas (28), Geobacter (2), Geopsychrobacter (10), Desulfuromonas (1), Desulfobulbus (9), Clostridium (27), Geothrix (3), Ochrobactrum (40), and Rhodopseudomonas (38), have been shown to produce electricity in an MFC. These bacteria have been grown on simple soluble substrates, such as glucose or acetate, that can be directly taken into the cell and used for energy production.Cellulose is the most abundant biopolymer in the world, and there is great interest in using this material as a substrate in an MFC. However, use of a particulate substrate in an MFC has not been well investigated. Cellulose must first be hydrolyzed to a soluble substrate that can be taken up by the cell. In previous MFC tests this has required the use of enzymes to hydrolyze the cellulose into sugars or the use of cocultures or mixed cultures (32, 33, 35). For example, Ren et al. (32) used a coculture of the cellulose fermentor Clostridium cellulolyticum and the exoelectrogen Geobacter sulfurreducens to generate electricity in an MFC fed with cellulose. Analysis of the anode microbial communities in other studies of cellulose-fed MFCs showed that Clostridium spp. (in a biofilm) and Comamonadaceae (in suspension) were predominant when rumen contents were used as an inoculum (35), while a rice paddy soil inoculum (12) converged to a Rhizobiales-dominated anode community (more than 30% of the population). To date, it has not been demonstrated that a single microbe can both degrade cellulose and generate current.Conventional methods of isolating exoelectrogenic microorganisms are based primarily on identifying microorganisms that can respire using soluble or insoluble metal oxides in agar plates (20-22). However, not all dissimilatory metal oxide-reducing bacteria are capable of producing electricity in an MFC, and not all bacteria that produce current in an MFC can grow using metal oxides (5, 34). Therefore, these methods may miss important electrochemically active strains of microorganisms. A new method to isolate exoelectrogenic microorganisms was recently developed (40); this method is based on dilution to extinction and a specially designed U-tube MFC that enriches exoelectrogenic bacteria on the anode. Using this method, a bacterium that could produce electricity in an MFC but not respire using iron was isolated (40).The main objective of this study was to isolate a bacterium capable of producing current from particulate cellulose. A cellulose-degrading consortium was diluted and serially transferred into U-tube MFCs using cellulose as the sole electron donor. Community analysis demonstrated the predominance of a single bacterium, which was isolated and compared to a culture collection strain for generation of current in an MFC.     

Measurement of opsonophagocytic activity of antibodies specific toNeisseria meningitidis serogroup A capsular polysaccharide-serogroup B outer membrane vesicle conjugate in animal model

Neisseria meningitidis is efficiently phagocytosed by polymorphonuclear leukocytes (PMNS) following opsonization with opsonic antibodies; opsonophagocytosis is the primary mechanism for clearance of meningococci from the host. Thus, in testing meningococcal vaccines, the level of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. Our previous studies demonstrated that the conjugation ofN. meningitidis serogroup A capsular polysaccharide (CPSA) to serogroup B outer membrane vesicle (OMV) could induce a high level of bactericidal antibody response against serogroup A meningococci in animals. The purpose of this study was to evaluate opsonophagocytic activity of the conjugate of CPSA to OMV (CPSA-OMV). In order to evaluate the potential efficacy of CPSA-OMV a flow cytometric opsonophagocytic assay was used. The conjugate and controls were injected intramuscularly into four groups of rabbits with boosters on days 14, 28 and 42 following primary immunization. The rabbits were bled prior to injection and two weeks after each injection. Opsonophagocytic activity of antibodies in hyperimmune sera through rabbit PMNS were measured with flow cytometer, using dihydrorhodamine-123 as a probe. The results indicated that our conjugate could induce a highly significant level of opsonophagocytic activity against serogroup A meningococci after 56 days compared to the control groups (P<0.05). We conclude that this conjugate represents a vaccine candidate against serogroups A and B meningococci after further investigation.     

Chemiluminescence determination of promazine in human serum and drug formulations using Ru(phen)32+–Ce(IV) system and a chemometrical optimization approach

A new chemiluminescence (CL) method using flow injection has been described for the rapid and sensitive determination of promazine hydrochloride (PMH). The method is based on the CL reaction of PMH with tris(1,10 phenanthroline)ruthenium(II), [Ru(phen)₃²⁺] and Ce(IV) in sulfuric acid medium. Effects of chemical variables were investigated employing central composite design and response surface methodology. Under the optimum conditions, the CL intensity was proportional to the concentration of the drug in solution over the ranges 0.020–0.32 and 0.32–32 µg/mL. The limit of detection (signal‐to‐noise ratio = 3) was 0.012 µg/mL. The method was applied successfully to the determination of PMH in drug formulations and human serum (recovery percentages between 96.7 and 105.0%). The relative standard deviation for 11 replicate determinations of 1.5 µg/mL of PMH was 1.7%. The minimum sampling rate was 100 samples per hour. Copyright © 2009 John Wiley & Sons, Ltd.     

The ENPP1 K121Q polymorphism is not associated with type 2 diabetes and related metabolic traits in an Iranian population

The K121Q polymorphism of the ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) gene has been variably associated with insulin resistance and type 2 diabetes (T2D) in several populations. However, this association has not been studied in Iranian subjects and we hypothesized that the K121Q variant might be associated with T2D and related metabolic traits in this population. The K121Q genotypes were determined by PCR-restriction fragment length polymorphism in 377 normoglycemic controls and 155 T2D patients. T2D patients had significantly higher values for systolic and diastolic blood pressure, BMI, glucose, cholesterol, triglyceride, LDL, apoB, insulin, and HOMA-IR, and lower levels of HDL than the normoglycemic subjects. The frequency of the Q allele did not differ between T2D and normoglycemic subjects (OR 0.96, 95% CI 0.90-2.00, P?=?0.70). The Q allele frequency was 16.5% in T2D and 15.2% in normoglycemic subjects. The ENPP1 genotype (KQ?+?QQ) was not associated with the systolic and diastolic blood pressure, glucose, triglyceride, cholesterol, LDL-C and HDL-C, apo B, BMI, HOMA-IR, and insulin levels in both normoglycemic and T2D groups. Our results suggest that the ENPP1 121Q allele might not be associated with T2D and related metabolic traits among Iranian subjects.     

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.